The sequencing of the human genome has allowed the identification of a huge number of putative genes.1,2,3 However, the function of only a small number of these can be inferred from their primary sequences. New techniques are needed to cope with the task of assigning functional roles to these gene products. This implies determination of how, when and where they are involved in specific biochemical pathways. Ideally, these methods will allow the rapid screening of substantial subsets of the sum of a genome's products. Although many proteins have been identified by functional cloning of novel genes, this ‘expression cloning’ approach remains a significant experimental challenge. Certain proteomic methods have been designed for broad and rapid screening, but they are largely limited to in vitro application and do not necessarily provide information pertinent to living cells. Moreover, although these methods can verify what genes are expressed, it is even more important to understand the dynamic patterns of in vivo protein expression and localization. For this, more powerful methods of detection of specific proteins and their interactions inside living cells are urgently required.
Several labeling techniques have been developed that involve the use of fluorescent dyes bearing reactive functional groups such as succinimidyl esters or maleimides, known to react with amines or thiols.4,5,6 Although these techniques are typically non-specific—many such functional groups exposed on the surface of any protein may be labeled—the characterization of these small molecule fluorophores teaches us the general requirements for solubility and cell permeability. However, in the proteomic context, they do not provide a general means for gathering information on specific protein targets.
The genetic fusion of target proteins to fluorescent proteins such as jellyfish green fluorescent protein (GFP) is another technique that has seen broad application.7 However, there are some serious limitations of this method. For example, the entire sequence of GFP must be properly folded into its 11-stranded β-barrel structure for it to function as a fluorophore, but it folds very slowly and is prone to aggregation. Moreover, GFP fluorescence suffers from low quantum yields, is sensitive to the environment of its fusion with test proteins and is also difficult to distinguish from the autofluorescent background of living cells. Furthermore, the steric bulk of a 27 kDa β-barrel protein can significantly perturb the interactions of the test proteins.7,8 In summary, the use of GFP derivatives can be inefficient and intrusive.
Of greater pertinence to this proposal is Tsien's use of certain small organometallic molecules that have been shown to react specifically with four cysteine residues.9,10,11 These cysteine residues were arranged in what was originally thought to be an α-helical conformation, but it was later shown that a β-turn conformation was optimal for their reaction with the fluorogenic arsenate compounds employed. In the application of this method, the fusion of a small probe protein of appropriate sequence to the target test protein allows it to be fluorescently labeled in live cells. Although these metallic complexes may not be broadly applicable to in vivo protein labeling studies due to their acute toxicity, they demonstrate nevertheless the feasibility of the use of small molecules to react preferentially with multiple thiol groups on a protein scaffold even in live cells, in the presence of several equivalents of simple native thiols. Furthermore, these small molecules illustrate the possibility the specific labeling of a test protein expressed as a fusion protein with a target sequence comprising an appropriate protein conformational motif. In this way, the seminal work of Tsien demonstrates the validity of our approach and the feasibility for our fluorometric assay proposed herein.
Over the last several years,13,14 we have conceived a rational design strategy parallel to Tsien's approach in which de novo, minimal peptides of less than 30 amino acids will react with novel synthetic probe reagents that fluoresce only after their reaction with the minimal folded peptide.